Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

Abstract

Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.Seen from a home economics point of view, making ribosomes is the main task of a living cell (36, 37). Synthesis of the ribosome constituents, their processing and assembly into mature particles, and the regulation of the entire process require the participation of numerous factors. Surprisingly, until relatively recently, few players involved in these processes had been identified. However, in the past decade, by means of genetic and biochemical tricks, and thanks to the tractability of the yeast Saccharomyces cerevisiae and, more recently, with in silico searches, dozens of factors (snoRNAs or protein factors) implicated in ribosome biogenesis have been characterized. For most of them, however, their molecular function in this process is not known.During the past year, systematic analyses of protein complexes in S. cerevisiae by tandem affinity chromatography purification (3,7,11,14,16) as well as proteomic analysis of the nucleolus of human cells (1) and definition of putative modular transcriptional networks by computer assisted analysis of the transcriptional expression patterns (18,20) led to a burst of new putative players. Through these approaches, new factors associated with the pre-60S particle, precursor of the large ribosomal subunit (LSU) and thus putatively involved in the maturation-assembly pathway of the LSU, have been identified. Likewise, factors associated with the early processing complexes containing the large 35S pre-rRNA have been characterized (11) and shown to include mostly proteins specifically required for small ribosomal subunit (SSU) production. In contrast, the nonribosomal proteins found in late nucleoprotein complex precursors of the SSU have not been ide...

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

“…The early 43S preribosomal particle is rapidly exported to the cytoplasm, where the conversion of 20S pre-rRNA into mature 18S rRNA and the last assembly reactions take place (Udem and Warner 1973). The study of the different purified pre-40S complexes indicates that only few transacting factors are needed to finish the maturation of 40S r-subunits from early 43S preribosomal particles (Schäfer et al 2002;Fatica et al 2003a;Vanrobays et al 2003). However, the study of the different purified pre-60S complexes is consistent with the presence of distinct pre-60S intermediates that move from the nucleolus to the nucleoplasm and from there to the cytoplasm.…”

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

“…Hence, the observed dynamic localization of Rio1 may reflect changes in its affinity for nucleolar factors and/or its active import-export from the nucleus. Rio1 shuttling between the nucleus and cytoplasm was evidenced previously by its intranuclear accumulation in an exportin mutant 16 .…”

“…To study Rio1 activity at the rDNA, we wished to inducibly remove the kinase from the nucleus only. Based on the observation that Rio1 shuttles between the nucleus and the cytoplasm 16 , we figured that conditionally removing its C-terminal tail containing a putative nuclear localization signal (NLS; Supplementary Fig. 5a) would prevent truncated Rio1 from entering the nucleus.…”

“…Hence, the observed dynamic localization of Rio1 may reflect changes in its affinity for nucleolar factors and/or its active import-export from the nucleus. Rio1 shuttling between the nucleus and cytoplasm was evidenced previously by its intranuclear accumulation in an exportin mutant 16 .Besides its localization, a role for Rio1 at the rDNA was indicated by its recent co-purification with the phosphatase Cdc14 and its inhibitor Cfi1 (ref. 23), which together with the histone deacetylase Sir2 form the rDNA-silencing complex RENT 24 (Fig.…”

“…Noteworthy, the RIO kinases may act especially as ATPases as they exhibit o0.1% kinase activity in vitro [12][13][14] . Cytoplasmic Rio1 contributes to pre-40S ribosome biogenesis by promoting 20S pre-rRNA maturation and by stimulating the recycling of trans-acting factors at the pre-40S subunit, both in yeast 12,[15][16][17][18] and human cells 19,20 . Roles in the nucleus are unknown for any RIO member, either in yeast or eukaryotes beyond.…”